Monoamine oxidases A and B (MAO A and B) are outer mitochondrial membrane-bound enzymes that function in the degradation of neurotransmitters such as serotonin, dopamine, and norepinephrine and have been pharmacologically important in the treatment of various pathological disorders such as depression, addition, and Parkinson's Disease. Each enzyme contains a covalent 8alpha-S-cysteinylFAD as a functional coenzyme. This project seeks continued support to determine the structure of each enzyme by x-ray crystallography to investigate the detailed mechanisms of H+ abstraction in catalysis. To facilitate these studies, we have developed methods for high level expression of each enzyme in Pichia pastrois. The role of the covalent FAD in MAO B will be investigated by disrupting the site for covalent FAD attachment by mutagenesis, followed by expression and purification of the mutant enzyme and comparison of its kinetic properties with WT enzyme The role of the C-terminal hydrophobic domain in enzyme structure and function for MAO A and B will be investigated in preparing the truncated forms and comparison of the purified mutants with WT enzyme properties. Initial studies are proposed to investigat3e the substrate specificity, inhibitor sensitivity, and structural properties of an expressed amine oxidase from Mycobacterium tuberculosis. Results from these studies should provide insights into the structures and mechanisms of these flavoprotein amine oxidases and lead to the development of new, specific drugs for the treatment of neuro-disorders and possibly tuberculosis infections.